Inter-laboratory comparison of routine autoantibody detection methods for autoimmune neuropathies and myasthenia gravis.

Serological tests are important to detect autoantibodies (autoAbs) in patients with autoimmune neuropathies (AN) and myasthenia gravis (MG) as they are biomarkers for diagnosis, stratification, treatment selection, and monitoring. However, tests to detect autoAbs frequently lack proper standardization and results differ across diagnostic laboratories. We compared results for tests routinely performed in Spanish diagnostic laboratories to detect AN and MG autoAbs. In the Spanish Society of Immunology Autoimmunity Group national workshop, serum samples from 13 patients with AN or MG were tested for anti-ganglioside, anti-myelin-associated glycoprotein (MAG), anti-nicotinic acetylcholine receptor (AChR), and anti-muscle-specific kinase (MuSK) autoAbs using reference methods and were distributed for analysis to 27 participating laboratories using their routine methods. Overserved were inter-laboratory variability and worryingly low sensitivity, especially for anti-ganglioside immunoglobulin G and anti-MAG autoAb detection. This pilot study reflects autoAbs detection state of the art in AN and MG testing in leading diagnostic laboratories in Spain, highlighting the need for standardization prior to clinical use.

Membrane Proteome-Wide Screening of Autoantibodies in CIDP Using Human Cell Microarray Technology.

BACKGROUND AND OBJECTIVES: Autoantibody discovery in complex autoimmune diseases is challenging. Diverse successful antigen identification strategies are available, but, so far, have often been unsuccessful, especially in the discovery of protein antigens in which conformational and post-translational modification are critical. Our study assesses the utility of a human membrane and secreted protein microarray technology to detect autoantibodies in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). METHODS: A cell microarray consisting of human embryonic kidney-293 cells expressing >5,000 human proteins was used. First, a validation step was performed with 4 serum samples from patients with autoimmune nodopathy (AN) to assess the ability of this technology to detect circulating known autoantibodies. The ability of the cell microarray technology to discover novel IgG autoantibodies was assessed incubating the array with 8 CIDP serum samples. Identified autoantibodies were subsequently validated using cell-based assays (CBAs), ELISA, and/or tissue immunohistochemistry and analyzed in a cohort of CIDP and AN (n = 96) and control (n = 100) samples. RESULTS: Serum anti-contactin-1 and anti-neurofascin-155 were detected by the human cell microarray technology. Nine potentially relevant antigens were found in patients with CIDP without other detectable antibodies; confirmation was possible in six of them: ephrin type-A receptor 7 (EPHA7); potassium-transporting ATPase alpha chain 1 and subunit beta (ATP4A/4B); leukemia-inhibitory factor (LIF); and interferon lambda 1, 2, and 3 (IFNL1, IFNL2, IFNL3). Anti-ATP4A/4B and anti-EPHA7 antibodies were detected in patients and controls and considered unrelated to CIDP. Both anti-LIF and anti-IFNL antibodies were found in the same 2 patients and were not detected in any control. Both patients showed the same staining pattern against myelinating fibers of peripheral nerve tissue and of myelinating neuron-Schwann cell cocultures. Clinically relevant correlations could not be established for anti-LIF and anti-IFNL3 antibodies. DISCUSSION: Our work demonstrates the utility of human cell microarray technology to detect known and discover unknown autoantibodies in human serum samples. Despite potential CIDP-associated autoantibodies (anti-LIF and anti-IFNL3) being identified, their clinical and pathogenic relevance needs to be elucidated in bigger cohorts.

Clinicopathological correlates in frontotemporal lobar degeneration: motor neuron disease spectrum.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease (MND) that shares a common clinical, genetic and pathologic spectrum with frontotemporal dementia (FTD). It is highly heterogeneous in its presentation and features. Up to 50% of patients with MND develop cognitive-behavioural symptoms during the course of the disease, meeting criteria for FTD in 10-15% of cases. In the absence of a precise biomarker, neuropathology is still a valuable tool to understand disease nosology, reach a definite diagnostic confirmation and help define specific subgroups of patients with common phenotypic, genetic and biomarker profiles. However, few neuropathological series have been published, and the frequency of FTLD in MND is difficult to estimate. In this work we describe a large clinicopathologic series of MND, analysing the frequency of concurrent FTLD changes and trying to define specific subgroups of patients based on their clinical, genetic and pathological characteristics. We performed an observational, retrospective, multi-centre case study. We included all cases meeting neuropathological criteria for MND from the Neurological Tissue Bank of the FRCB-IDIBAPS-Hospital Clínic Barcelona Biobank between 1994 and 2022, regardless of their last clinical diagnosis. While brain donation is encouraged in all patients, it is performed in very few, and representativeness of the cohort might not be precise for all patients with MND. We retrospectively reviewed clinical and neuropathological data, and describe the main clinical, genetic and pathogenic features, comparing neuropathologic groups between MND with and without FTLD changes and aiming to define specific subgroups. We included brain samples from 124 patients, 44 of whom (35.5%) had FTLD neuropathologic features (i.e. FTLD-MND). Pathologic TDP-43 aggregates were present in 93.6% of the cohort and were more extensive (higher Brettschneider stage) in those with concurrent FTLD (p < 0.001). Motor symptom onset was more frequent in the bulbar region in FTLD-MND cases than in those with isolated MND (p = 0.023), with no differences in survival. We observed a better clinicopathological correlation in the MND group than in the FTLD-MND group (93.8% vs 61.4%; p < 0.001). Pathogenic genetic variants were more common in the FTLD-MND group, especially C9orf72. We describe a frequency of FTLD of 35.5% in our series of neuropathologically confirmed cases of MND. The FTLD-MND spectrum is highly heterogeneous in all aspects, especially in patients with FTLD, in whom it is particularly difficult to define specific subgroups. In the absence of definite biomarkers, neuropathology remains a valuable tool for a definite diagnosis, increasing our knowledge in disease nosology.

Refocusing generalized myasthenia gravis: Patient burden, disease profiles, and the role of evolving therapy.

BACKGROUND AND PURPOSE: Generalized myasthenia gravis (gMG) continues to present significant challenges for clinical management due to an unpredictable disease course, frequent disease fluctuations, and varying response to therapy. The recent availability of new pharmacologic therapies presents a valuable opportunity to reevaluate how this disease is classified, assessed, and managed and identify new ways to improve the clinical care of patients with gMG. METHODS: Narrative review was made of publications identified via searches of PubMed and selected congresses (January 2000-September 2022). RESULTS: New consensus definitions are required to ensure consistency, to better characterize patients, and to identify patients who will benefit from specific drugs and earlier use of these agents. There is a need for more frequent, standardized patient assessment to identify the cause of motor function deficits, provide a clearer picture of the disease burden and its impact on daily living and quality of life (QoL), and better support treatment decision-making. Novel approaches that target different components of the immune system will play a role in more precise treatment of patients with gMG, alongside the development of new algorithms to guide individualized patient management. CONCLUSIONS: gMG has a physical, mental, and social impact, resulting in a considerable burden of disease and substantially decreased QoL, despite standard treatments. The availability of novel, targeted treatments that influence key pathological mediators of gMG, together with new biomarkers, offers the potential to optimize patient management and ultimately enables a greater number of patients to achieve minimal manifestation status and a reduced burden of disease.

Rituximab treatment in myasthenia gravis.

Myasthenia gravis (MG) is a chronic autoimmune disease mediated by antibodies against post-synaptic proteins of the neuromuscular junction. Up to 10%-30% of patients are refractory to conventional treatments. For these patients, rituximab has been used off-label in the recent decades. Rituximab is a monoclonal antibody against the CD20 protein that leads to B cell depletion and to the synthesis of new antibody-secreting plasma cells. Although rituximab was created to treat B-cell lymphoma, its use has widely increased to treat autoimmune diseases. In MG, the benefit of rituximab treatment in MuSK-positive patients seems clear, but a high variability in the results of observational studies and even clinical trials has been reported for AChR-positive patients. Moreover, few evidence has been reported in seronegative MG and juvenile MG and some questions about regimen of administration or monitoring strategies, remains open. In this review, we intend to revise the available literature on this topic and resume the current evidence of effectiveness of Rituximab in MG, with special attention to results on every MG subtype, as well as the administration protocols, monitoring strategies and safety profile of the drug.

Antibodies against the flotillin-1/2 complex in patients with multiple sclerosis.

Multiple sclerosis is a tissue-specific autoimmune disease of the central nervous system in which the antigen(s) remains elusive. Antibodies targeting the flotillin-1/2 complex have been described in 1-2% of the patients in a recent study. Other candidate antigens as anoctamin-2 or neurofascin-155 have been previously described in multiple sclerosis patients, although their clinical relevance remains uncertain. Our study aims to analyse the frequency and clinical relevance of antibodies against neurofascin-155, anoctamin-2 and flotillin-1/2 complex in multiple sclerosis. Serum (n = 252) and CSF (n = 50) samples from 282 multiple sclerosis patients were included in the study. The control group was composed of 260 serum samples (71 healthy donors and 189 with other neuroinflammatory disorders). Anti-flotillin-1/2, anti-anoctamin-2 and anti-neurofascin-155 antibodies were tested by cell-based assays using transfected cells. We identified six multiple sclerosis patients with antibodies against the flotillin-1/2 complex (2.1%) and one multiple sclerosis patient with antibodies against anoctamin-2 (0.35%). All multiple sclerosis patients were negative for anti-neurofascin-155 antibodies. Three of the anti-flotillin-1/2 positive patients showed anti-flotillin-1/2 positivity in other serum samples extracted at different moments of their disease. Immunoglobulin G subclasses of anti-flotillin-1/2 antibodies were predominantly one and three. We confirm that antibodies targeting the flotillin-1/2 complex are present in a subgroup of patients with multiple sclerosis. Further studies are needed to understand the clinical and pathological relevance of anti-flotillin-1/2 autoantibodies in multiple sclerosis.

Reduced Number of Thymoma CTLA4-Positive Cells Is Associated With a Higher Probability of Developing Myasthenia Gravis.

BACKGROUND AND OBJECTIVES: Myasthenia gravis (MG) is an autoimmune disease associated with comorbid thymoma in 10%-15% of cases. Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) expressed by T cells downregulates T-cell-mediated immune response. Polymorphisms in the CTLA4 gene have been associated with the development of MG. In this context, we aimed to determine whether CTLA4 expression in the thymoma differs between patients with and without MG and whether CTLA4 gene polymorphisms are associated with these differences. METHODS: This is a retrospective study of all patients, with and without MG, surgically treated at our institution for thymoma between January 2010 and December 2020. Ten samples were obtained from normal thymuses as controls. The number of CTLA4-positive cells in paraffin-embedded thymoma samples was determined by immunohistochemistry. The presence of follicular-center and regulatory T-cell lymphocytes was determined by immunohistochemistry (B-cell lymphoma [BCL]-6 expression) and double immunofluorescence-based staining of CD4-FOXP3, respectively. We evaluated the association between thymic expression of CTLA4 and the development of MG. We also determined the association between CTLA4 expression and various clinical and prognostic characteristics of MG. We sequenced the CTLA4 gene and evaluated possible associations between CTLA4 polymorphisms and thymic CTLA4 expression. Finally, we assessed the potential association between these polymorphisms and the risk of MG. RESULTS: Forty-one patients with thymoma were included. Of them, 23 had comorbid MG (56.1%). On average, patients with MG had fewer CTLA4-positive cells in the thymoma than non-MG patients: 69.3 cells/mm2 (95% CIs: 39.6-99.1) vs 674.4 (276.0-1,024.0) cells/mm2; p = 0.001 and vs controls (200.74 [57.9-343.6] cells/mm2; p = 0.02). No between-group differences (MG vs non-MG) were observed in the number of cells positive for BCL6 or CD4-FOXP3. CTLA4 expression was not associated with differences in MG outcome or treatment refractoriness. Two polymorphisms were detected in the CTLA4 gene, rs231770 (n = 30 patients) and rs231775 (n = 17). MG was present in a similar proportion of patients for all genotypes. However, a nonsignificant trend toward a lower CTLA4-positive cell count was observed among carriers of the rs231775 polymorphism vs noncarriers: 77.9 cells/mm2 (95% CI: -51.5 to 207.5) vs 343.3 cells/mm2 (95% CI: 126.2-560.4). DISCUSSION: Reduced CTLA4 expression in thymoma may predispose to a higher risk of developing MG.

Leptospirosis-induced acute acquired inflammatory neuropathy.

Leptospirosis is a zoonotic infection that can present with neurological manifestations. Although uncommon, it may affect the peripheral nervous system in the form of polyradiculoneuropathy. We report the case of a 30-year-old male who developed flaccid tetraparesis and multiple cranial neuropathies on the fourteenth day of admission to the intensive care unit for fever and multi-organ failure. We also review the existing literature about peripheral nerve damage in leptospirosis and present our hypothesis on the possible pathogenic mechanisms. Electrophysiological findings were consistent with acute demyelinating polyradiculoneuropathy and extensive blood tests were positive for leptospiral IgM and IgG antibodies. Treatment with plasmapheresis was begun, followed by intravenous immunoglobulin (IVIg), and the patient improved slowly. Our work adds to the evidence of leptospirosis infection as a cause of acute demyelinating polyneuropathy. The possibility that leptospirosis-polyradiculoneuropathy may be caused by an immune pathogenesis emphasizes the importance of identifying this entity because immunomodulatory therapy could play a vital role in the recovery process.

Neuroinflammation-Related Proteins NOD2 and Spp1 Are Abnormally Upregulated in Amyotrophic Lateral Sclerosis.

BACKGROUND AND OBJECTIVES: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown etiology and poorly understood pathophysiology. There is no specific biomarker either for diagnosis or prognosis. The aim of our study was to investigate differentially expressed proteins in the CSF and serum from patients with ALS to determine their role in the disease process and evaluate their utility as diagnostic or prognostic biomarkers. METHODS: We performed mass spectrometry in the CSF from 3 patients with ALS and 3 healthy controls (HCs). The results were compared with motor cortex dysregulated transcripts obtained from 11patients with sporadic ALS and 8 HCs. Candidate proteins were tested using ELISA in the serum of 123 patients with ALS, 30 patients with Alzheimer disease (AD), 28 patients with frontotemporal dementia (FTD), and 102 HCs. Patients with ALS, AD, and FTD were prospectively recruited from January 2003 to December 2020. A group of age-matched HCs was randomly selected from the Sant Pau Initiative on Neurodegeneration cohort of the Sant Pau Memory Unit. RESULTS: Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and osteopontin (Spp1) were differentially expressed in the CSF and the motor cortex transcriptome of patients with ALS compared with that in HCs (p < 0.05). NOD2 and Spp1 levels were significantly higher in sera from patients with ALS than in HCs (p < 0.001). Receiver operating characteristic analysis showed an area under the curve of 0.63 for NOD2 and 0.81 for Spp1. NOD2 levels were significantly lower in patients with AD and FTD than in patients with ALS (p < 0.0001), but we found no significant differences in Spp1 levels between patients with ALS, AD (p = 0.51), and FTD (p = 0.42). We found a negative correlation between Spp1 levels and ALS functional rating scale (r = -0.24, p = 0.009). DISCUSSION: Our discovery-based approach identified NOD2 as a novel biomarker in ALS and adds evidence to the contribution of Spp1 in the disease process. Both proteins are involved in innate immunity and autophagy and are increased in the serum from patients with ALS. Our data support a relevant role of neuroinflammation in the pathophysiology of the disease and may identify targets for disease-modifying treatments in ALS. Further longitudinal studies should investigate the diagnostic and prognostic value of NOD2 and Spp1 in clinical practice.

Azathioprine therapy induces selective NK cell depletion and IFN-γ deficiency predisposing to herpesvirus reactivation.

BACKGROUND: Azathioprine is a widely prescribed drug for patients with chronic inflammatory diseases such as myasthenia gravis or organ transplant recipients. Azathioprine exerts immunosuppressive effects by inhibiting intracellular purine synthesis and reducing the numbers of circulating B and T lymphocytes. Case reports indicate increased risk for serious infections that can occur despite regular measurements of lymphocyte counts during azathioprine therapy. OBJECTIVE: We sought to comprehensively investigate therapy-associated patient risks and the underlying immune dysfunction of azathioprine use. METHODS: Peripheral blood leukocytes were analyzed using single-cell mass and spectral flow cytometry to detect specific effects of azathioprine use on the systemic immune signature. Therapy-associated clinical features were analyzed in 2 independent cohorts of myasthenia gravis patients. RESULTS: Azathioprine therapy selectively induced pronounced CD56dimCD16+ natural killer cell depletion and concomitant IFN-γ deficiency. Cytokine profiling revealed a specific contraction of classical TH1 cells during azathioprine treatment. We further observed an increased occurrence of reactivation of endogenous latent herpesviruses in the azathioprine-treated group versus in patients with myasthenia gravis who were not receiving immunomodulatory treatment; this increased occurrence was validated in an independent cohort. CONCLUSION: Our study highlights the risk of development of adverse events during azathioprine therapy and suggests that natural killer cell monitoring could be valuable in clinical practice.

Rituximab in myasthenia gravis: efficacy, associated infections and risk of induced hypogammaglobulinemia.

The aim of this study is to evaluate the long-term efficacy, safety, and impact on immunoglobulin G (IgG) levels of rituximab in patients with myasthenia gravis (MG). A retrospective, observational study of drug-refractory MG patients treated with rituximab was done. The MG Foundation of America postintervention status (MGFA-PIS) was used to evaluate clinical response. Serum IgG levels were determined at baseline and post-treatment. Hypogammaglobulinemia was defined as IgG<7g/L. Thirty patients were included, 12 with anti-MuSK and 18 with anti-AChR antibodies. Mean (SD) follow-up was 85.5 (48) months. All 12 MuSK+ patients but only six (33%) AChR+ patients achieved minimal manifestations or remission (p<0.01). Nine severe infections were observed in five patients (17%). One patient was diagnosed with progressive multifocal leukoencephalopathy. At baseline, two patients (2/24; 8%) had hypogammaglobulinemia. During follow-up, hypogammaglobulinemia was observed in 60% (3/5) of patients who developed an infection and in 33% (7/21) who did not. Two of these patients died of infection-related complications. This study supports the effectiveness of rituximab in patients with MG, especially those with anti-MuSK antibodies. Severe infections may appear after rituximab treatment and hypogammaglobulinemia might play a role on it. A standard protocol would be needed to closely monitor IgG levels in MG patients treated with rituximab.

Re-programming mouse liver-resident invariant natural killer T cells for suppressing hepatic and diabetogenic autoimmunity.

Invariant NKT (iNKT) cells comprise a heterogeneous group of non-circulating, tissue-resident T lymphocytes that recognize glycolipids, including alpha-galactosylceramide (αGalCer), in the context of CD1d, but whether peripheral iNKT cell subsets are terminally differentiated remains unclear. Here we show that mouse and human liver-resident αGalCer/CD1d-binding iNKTs largely correspond to a novel Zbtb16+Tbx21+Gata3+MaflowRorc- subset that exhibits profound transcriptional, phenotypic and functional plasticity. Repetitive in vivo encounters of these liver iNKT (LiNKT) cells with intravenously delivered αGalCer/CD1d-coated nanoparticles (NP) trigger their differentiation into immunoregulatory, IL-10+IL-21-producing Zbtb16highMafhighTbx21+Gata3+Rorc- cells, termed LiNKTR1, expressing a T regulatory type 1 (TR1)-like transcriptional signature. This response is LiNKT-specific, since neither lung nor splenic tissue-resident iNKT cells from αGalCer/CD1d-NP-treated mice produce IL-10 or IL-21. Additionally, these LiNKTR1 cells suppress autoantigen presentation, and recognize CD1d expressed on conventional B cells to induce IL-10+IL-35-producing regulatory B (Breg) cells, leading to the suppression of liver and pancreas autoimmunity. Our results thus suggest that LiNKT cells are plastic for further functional diversification, with such plasticity potentially targetable for suppressing tissue-specific inflammatory phenomena.

Immune Response and Safety of SARS-CoV-2 mRNA-1273 Vaccine in Patients With Myasthenia Gravis.

BACKGROUND AND OBJECTIVES: Evidence regarding the safety and efficacy of messenger RNA (mRNA) vaccines in patients with myasthenia gravis (MG) after immunosuppressive therapies is scarce. Our aim is to determine whether the mRNA-1273 vaccine is safe and able to induce humoral and cellular responses in patients with MG. METHODS: We performed an observational, longitudinal, prospective study including 100 patients with MG of a referral center for MG in our country, conducted from April 2021 to November 2021 during the vaccination campaign. The mRNA-1273 vaccine was scheduled for all participants. Blood samples were collected before vaccination and 3 months after a second dose. Clinical changes in MG were measured using the MG activities of daily life score at baseline and 1 week after the first and second doses. A surveillance of all symptoms of coronavirus disease 2019 (COVID-19) was conducted throughout the study. Humoral and cellular immune responses after vaccination were assessed using a spike-antibody ELISA and interferon gamma release assay in plasma. The primary outcomes were clinically significant changes in MG symptoms after vaccination, adverse events (AEs), and seroconversion and T-cell immune response rates. RESULTS: Ninety-nine patients completed the full vaccination schedule, and 98 had 2 blood samples taken. A statistically significant worsening of symptoms was identified after the first and second doses of the mRNA-1273 vaccine, but this was not clinically relevant. Mild AEs occurred in 14 patients after the first dose and in 21 patients after the second dose. Eighty-seven patients developed a humoral response and 72 patients showed a T-cell response after vaccination. A combined therapy with prednisone and other immunosuppressive drugs correlated with a lower seroconversion ratio (OR = 5.97, 95% CI 1.46-24.09, p = 0.015) and a lower T-cell response ratio (OR = 2.83, 95% CI 1.13-7.13, p = 0.024). DISCUSSION: Our findings indicate that the mRNA vaccination against COVID-19 is safe in patients with MG and show no negative impact on the disease course. Patients achieved high humoral and cellular immune response levels. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that patients with MG receiving the mRNA-1273 vaccine did not show clinical worsening after vaccination and that most of the patients achieved high cellular or immune response levels.

Drug-refractory myasthenia gravis: Clinical characteristics, treatments, and outcome.

OBJECTIVE: To describe the clinical characteristics and outcomes in patients with refractory myasthenia gravis (MG) and to determine the effectiveness and side effects of the drugs used for their treatment. METHODS: This observational retrospective cross-sectional multicenter study was based on data from the Spanish MG Registry (NMD-ES). Patients were considered refractory when their MG Foundation of America post-interventional status (MGFA-PIS) was unchanged or worse after corticosteroids and two or more other immunosuppressive agents. Clinical and immunologic characteristics of drug-refractory patients, efficiency and toxicity of drugs used, and outcome (MGFA-PIS) at end of follow-up were studied. RESULTS: We included 990 patients from 15 hospitals. Eighty-four patients (68 of 842 anti-acetylcholine receptor [AChR], 5 of 26 anti-muscle-specific tyrosine kinase [MusK], 10 of 120 seronegative, and 1 of 2 double-seropositive patients) were drug refractory. Drug-refractory patients were more frequently women (p < 0.0001), younger at onset (p < 0.0001), and anti-MuSK positive (p = 0.037). Moreover, they more frequently presented a generalized form of the disease, bulbar symptoms, and life-threatening events (p < 0.0001; p = 0.018; and p = 0.002, respectively) than non-drug-refractory patients. Mean follow-up was 9.8 years (SD 4.5). Twenty-four (50%) refractory patients had side effects to one or more of the drugs. At the end of follow-up, 42.9% of drug-refractory patients (42.6% of anti-AChR, 100% of anti-MuSK, and 10% of seronegative patients) and 79.8% of non-drug-refractory patients (p < 0.0001) achieved remission or had minimal manifestations. Eighty percent of drug-refractory-seronegative patients did not respond to any drug tested. INTERPRETATION: In this study, 8.5% of MG patients were drug-refractory. New more specific drugs are needed to treat drug-refractory MG patients.

Clinical and Laboratory Features in Anti-NF155 Autoimmune Nodopathy.

BACKGROUND AND OBJECTIVES: To study the clinical and laboratory features of antineurofascin-155 (NF155)-positive autoimmune nodopathy (AN). METHODS: Patients with anti-NF155 antibodies detected on routine immunologic testing were included. Clinical characteristics, treatment response, and functional scales (modified Rankin Scale [mRS] and Inflammatory Rasch-built Overall Disability Scale [I-RODS]) were retrospectively collected at baseline and at the follow-up. Autoantibody and neurofilament light (NfL) chain levels were analyzed at baseline and at the follow-up. RESULTS: Forty NF155+ patients with AN were included. Mean age at onset was 42.4 years. Patients presented with a progressive (75%), sensory motor (87.5%), and symmetric distal-predominant weakness in upper (97.2%) and lower extremities (94.5%), with tremor and ataxia (75%). Patients received a median of 3 (2-4) different treatments in 46 months of median follow-up. Response to IV immunoglobulin (86.8%) or steroids (72.2%) was poor in most patients, whereas 77.3% responded to rituximab. HLA-DRB1*15 was detected in 91.3% of patients. IgG4 anti-NF155 antibodies were predominant in all patients; anti-NF155 titers correlated with mRS within the same patient (r = 0.41, p = 0.004). Serum NfL (sNfL) levels were higher in anti-NF155+ AN than in healthy controls (36.47 vs 7.56 pg/mL, p < 0.001) and correlated with anti-NF155 titers (r = 0.43, p = 0.001), with I-RODS at baseline (r = -0.88, p < 0.001) and with maximum I-RODS achieved (r = -0.58, p = 0.01). Anti-NF155 titers and sNfL levels decreased in all rituximab-treated patients. DISCUSSION: Anti-NF155 AN presents a distinct clinical profile and good response to rituximab. Autoantibody titers and sNfL are useful to monitor disease status in these patients. The use of untagged-NF155 plasmids minimizes the detection of false anti-NF155+ cases. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that anti-NF155 antibodies associate with a specific phenotype and response to rituximab.

Autoantibody screening in Guillain-Barré syndrome.

BACKGROUND: Guillain-Barré syndrome (GBS) is an acute inflammatory neuropathy with a heterogeneous presentation. Although some evidences support the role of autoantibodies in its pathogenesis, the target antigens remain unknown in a substantial proportion of GBS patients. The objective of this study is to screen for autoantibodies targeting peripheral nerve components in Guillain-Barré syndrome. METHODS: Autoantibody screening was performed in serum samples from all GBS patients included in the International GBS Outcome study by 11 different Spanish centres. The screening included testing for anti-ganglioside antibodies, anti-nodo/paranodal antibodies, immunocytochemistry on neuroblastoma-derived human motor neurons and murine dorsal root ganglia (DRG) neurons, and immunohistochemistry on monkey peripheral nerve sections. We analysed the staining patterns of patients and controls. The prognostic value of anti-ganglioside antibodies was also analysed. RESULTS: None of the GBS patients (n = 100) reacted against the nodo/paranodal proteins tested, and 61 (61%) were positive for, at least, one anti-ganglioside antibody. GBS sera reacted strongly against DRG neurons more frequently than controls both with IgG (6% vs 0%; p = 0.03) and IgM (11% vs 2.2%; p = 0.02) immunodetection. No differences were observed in the proportion of patients reacting against neuroblastoma-derived human motor neurons. Reactivity against monkey nerve tissue was frequently detected both in patients and controls, but specific patterns were only detected in GBS patients: IgG from 13 (13%) patients reacted strongly against Schwann cells. Finally, we confirmed that IgG anti-GM1 antibodies are associated with poorer outcomes independently of other known prognostic factors. CONCLUSION: Our study confirms that (1) GBS patients display a heterogeneous repertoire of autoantibodies targeting nerve cells and structures; (2) gangliosides are the most frequent antigens in GBS patients and have a prognostic value; (3) further antigen-discovery experiments may elucidate other potential antigens in GBS.

Clinical characteristics and outcomes of thymoma-associated myasthenia gravis.

BACKGROUND AND PURPOSE: Prognosis of myasthenia gravis (MG) in patients with thymoma is not well established. Moreover, it is not clear whether thymoma recurrence or unresectable lesions entail a worse prognosis of MG. METHODS: This multicenter study was based on data from a Spanish neurologist-driven MG registry. All patients were aged >18 years at onset and had anti-acetylcholine receptor antibodies. We compared the clinical data of thymomatous and nonthymomatous patients. Prognosis of patients with recurrent or nonresectable thymomas was assessed. RESULTS: We included 964 patients from 15 hospitals; 148 (15.4%) had thymoma-associated MG. Median follow-up time was 4.6 years. At onset, thymoma-associated MG patients were younger (52.0 vs. 60.4 years, p < 0.001), had more generalized symptoms (odds ratio [OR]: 3.02, 95% confidence interval [CI]: 1.95-4.68, p < 0.001) and more severe clinical forms according to the Myasthenia Gravis Foundation of America (MGFA) scale (OR: 1.6, 95% CI: 1.15-2.21, p = 0.005). Disease severity based on MGFA postintervention status (MGFA-PIS) was higher in thymomatous patients at 1 year, 5 years, and the end of follow-up. Treatment refractoriness and mortality were also higher (OR: 2.28, 95% CI: 1.43-3.63, p = 0.001; hazard ratio: 2.46, 95% CI: 1.47-4.14, p = 0.001). Myasthenic symptoms worsened in 13 of 27 patients with recurrences, but differences in long-term severity were not significant. Fifteen thymomatous patients had nonresectable thymomas with worse MGFA-PIS and higher mortality at the end of follow-up. CONCLUSIONS: Thymoma-associated MG patients had more severe myasthenic symptoms and worse prognosis. Thymoma recurrence was frequently associated with transient worsening of MG, but long-term prognosis did not differ from nonrecurrent thymoma. Patients with nonresectable thymoma tended to present severe forms of MG.

Pathophysiological Underpinnings of Extra-Motor Neurodegeneration in Amyotrophic Lateral Sclerosis: New Insights From Biomarker Studies.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) lie at opposing ends of a clinical, genetic, and neuropathological continuum. In the last decade, it has become clear that cognitive and behavioral changes in patients with ALS are more frequent than previously recognized. Significantly, these non-motor features can impact the diagnosis, prognosis, and management of ALS. Partially overlapping neuropathological staging systems have been proposed to describe the distribution of TAR DNA-binding protein 43 (TDP-43) aggregates outside the corticospinal tract. However, the relationship between TDP-43 inclusions and neurodegeneration is not absolute and other pathophysiological processes, such as neuroinflammation (with a prominent role of microglia), cortical hyperexcitability, and synaptic dysfunction also play a central role in ALS pathophysiology. In the last decade, imaging and biofluid biomarker studies have revealed important insights into the pathophysiological underpinnings of extra-motor neurodegeneration in the ALS-FTLD continuum. In this review, we first summarize the clinical and pathophysiological correlates of extra-motor neurodegeneration in ALS. Next, we discuss the diagnostic and prognostic value of biomarkers in ALS and their potential to characterize extra-motor neurodegeneration. Finally, we debate about how biomarkers could improve the diagnosis and classification of ALS. Emerging imaging biomarkers of extra-motor neurodegeneration that enable the monitoring of disease progression are particularly promising. In addition, a growing arsenal of biofluid biomarkers linked to neurodegeneration and neuroinflammation are improving the diagnostic accuracy and identification of patients with a faster progression rate. The development and validation of biomarkers that detect the pathological aggregates of TDP-43 in vivo are notably expected to further elucidate the pathophysiological underpinnings of extra-motor neurodegeneration in ALS. Novel biomarkers tracking the different aspects of ALS pathophysiology are paving the way to precision medicine approaches in the ALS-FTLD continuum. These are essential steps to improve the diagnosis and staging of ALS and the design of clinical trials testing novel disease-modifying treatments.